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Abstract

Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

AAV DNA replication is carried out by a rolling hairpin mechanism in cells co-infected by AAV and helper viruses such as adenovirus or herpes virus (Ward, 2006). The AAV DNA consists of a 4.7 kb linear DNA molecule with inverted terminal repeats (ITRs) that fold back to form T-shaped hairpin structures. The 3’ end hairpin serves as a primer for the replication of the AAV DNA. These hairpin structures are regenerated by the AAV Rep protein, allowing further rounds of replication (Im and Muzyczka, 1990). Both + and - strands of the AAV DNA are packaged and are infectious (Rose et al., 1969). When replicating AAV DNA is analyzed, several replicative intermediates can be detected (Straus et al., 1976). The most abundant replicative intermediate is a linear monomeric duplex molecule, formed by one + and one - strand of the AAV DNA, which is thought to be the immediate precursor of progeny single-stranded molecules that will be packaged in pre-formed capsids (Straus et al., 1976). Dimeric replicative intermediates are also common, and the AAV replication model is compatible with even larger replicative intermediates. The study of AAV replication benefitted from the discovery that AAV plasmids are infectious–the AAV DNA can be fully rescued from a plasmid (in the presence of helper virus) and its replication mimics that of the native virus (Samulski et al., 1982). The method detailed here allows the investigation of the DNA intermediates formed during DNA replication initiated from an AAV plasmid, and was used to compare different mutants of the AAV Rep protein for their ability to support AAV replication. The same method can be used to study other aspects of the AAV life cycle that can affect DNA replication of this virus, such as the effect of helper virus proteins or other factors that restrict/enhance AAV replication.

Seed 4.3 x 106 293T cells in a 10 cm dish in 10 ml of growth medium (DMEM + 10% FBS) for transfection next day. Include an additional plate to determine cell number at the time of infection with adenovirus. Cells are maintained for 24 h at 37 °C and 5% CO2.

Dilute 94 µl of PEI in 500 µl of SF DMEM and gently mix. The transfection reagents should be at room temperature (RT) before mixing. Incubate 5 min at RT.

Combine the PEI and DNA and gently mix by pipetting. Incubate at RT for 20 min.

Add the mixture to the medium in which the cells are growing. Pipet carefully to avoid detaching the cells.

4 h after transfection, determine the cell number (for example using a hemocytometer) in the extra plate.

Infect the cells using 5 pfu/cell of adenovirus. Include a condition without adenovirus as a negative control for AAV replication.

40 to 50 h post transfection a cytopathogenic effect is expected. Cells will round up and eventually detach (Figure 1).Note: The amount of adenovirus necessary to fully support AAV replication and to cause a cytopathogenic effect at 40 to 50 h post transfection can vary depending on the health of the cell line used and its passage number. This may need to be optimized before starting this protocol: infect cells with increasing amounts of adenovirus and monitor the appearance of a cytopathogenic effect (Figure 1).

Figure 1. AAV and adenovirus induced cytopathogenic effect. Left panel: 24 h post-transfection cells are adherent and form a monolayer. Right panel: 55 h post-transfection the cytopathogenic is evident; cells have rounded up and are lifting from the plate. Scale bars = 50 µm.

Extraction of low molecular weight

Collect cells and supernatant in a 15 ml conical tube. Cells should be loosely adherent and can be detached by pipetting. Wash the plate with 2 ml 1x DPBS and add to the 15 ml tube.

Centrifuge for 5 min at 500 x g at RT.

Discard the supernatant, and resuspend the cell pellet in 1 ml DPBS. Transfer 250 µl to a new 1.5 ml tube. The remainder of the cells can be harvested for additional analyses, e.g., gene and protein expression.

Centrifuge for 5 min at 500 x g at RT in a benchtop centrifuge and discard supernatant.

Add 500 µl of Hirt lysis buffer and mix by flicking or inverting the tube. Note: The lysate will be very viscous and mixing by pipetting will create bubbles.

Carefully transfer the supernatant (approximately 500 µl) to a new 1.5 ml tube. Note: If the supernatant cannot be separated sufficiently from the viscous pellet, a longer centrifugation may be required.

Under a fume hood, add 1 volume (500 µl) of phenol/chloroform/isoamyl alcohol and mix by inverting the tube until homogeneous.

Carefully remove and discard the supernatant, then wash the DNA pellet with 100 µl of 70% ethanol.

Centrifuge for 10 min at 17,000 x g at 4 °C.

Remove supernatant and air-dry the DNA pellet.

Resuspend the pellet in 80 µl ddH2O.

Determine the concentration of DNA in the samples using a NanoDrop spectrophotometer. Typical DNA concentration is around 750 ng/µl.

Southern blot assay (gel transfer)

Digest approximately 1 µg of the extracted DNA with DpnI, for 2 h at 37 °C. DpnI specifically digests methylated DNA, e.g., DNA amplified in dam+ bacteria strains. This step ensures that the input plasmid DNA is digested while newly replicated AAV DNA will not be affected.

Separate the digested DNA on a 0.8% agarose gel overnight at 1 V/cm.

Remove the gel from the electrophoresis tank and incubate for 30 min in denaturing solution on a platform shaker at approximately 25 rpm.

Rinse the gel twice in ddH2O.

Incubate the gel for 30 min in neutralizing solution on a platform shaker at approximately 25 rpm.

Replace the neutralizing solution with fresh neutralizing solution and incubate for a further 30 min while shaking.

Rinse in ddH2O, and then proceed with the assembly of the transfer set-up as detailed in Figure 2.

Figure 2. Scheme of capillary transfer method for Southern blotting. The support plate is a glass plate resting on the sides of the glass tray containing the 20x SSC. The paper towels layer is approximately 15 cm high. The weight used was approximately 600 g.

Incubate overnight to transfer the DNA in the gel to the nylon membrane by the capillary method (Figure 2). To obtain the same sample orientation on the nylon membrane as on the gel, turn the gel upside down.

Disassemble the set-up and carefully remove the membrane.

Rinse the membrane in 2x SSC, and let it air-dry.

To fix the DNA to the membrane, UV cross-link the membrane with 120,000 µJ/cm2.

The template to prepare the Rep and Amp specific probes is obtained by PCR on the pAV2 plasmid using the aforementioned primer pairs and the PCR conditions listed below. The PCR products are gel-purified and quantified using a NanoDrop spectrophotometer. The expected size of the PCR products is 338 base-pairs for the Rep PCR and 587 for the Amp PCR. 20 to 25 ng of purified PCR product DNA is used as DNA template to prepare the probes.
For both Rep and Amp PCRs, mix in a final volume of 25 µl:

Expose the membrane to the Phosphoimager screen for at least 2 h. Note: The length of exposure necessary to obtain the best signal can vary depending on the strength of the signal.

Acquire images using the Typhoon phosphor imager. Image analysis can be performed using the ImageQuant analysis software (GE Healthcare).

Data analysis

The gel images acquired following the procedure described above were analyzed using the ImageQuant analysis software (GE Healthcare). The level of AAV replication under different conditions is proportional to the intensity of the bands corresponding to viral replicative intermediates in DpnI-treated samples hybridized with the viral (Rep) probe (Figure 3). For wt AAV2 virus, monomeric and dimeric molecules of approximately 4.7 kb and 9.4 kb, respectively should be visible. Additional higher molecular weight intermediates can also be present. Membranes hybridized with the Amp probe allow for the assessment of efficiency of transfection and efficacy of DpnI digestion (Figure 3). The ImageQuant software supports band quantification by densitometry if a quantitative band comparison is required.

Figure 3. Analysis of AAV replication intermediates by Southern blot. Left panel: pAAV-GFP (no Rep control), pAV2 (WT AAV plasmid) and pAV2-RepK340H (replication-deficient Rep mutant) were transfected in the presence (+ Ad) or absence of adenovirus. The Amp probe binds to all plasmids containing the ampicillin resistance gene. The Rep probe only binds to DNA containing the AAV Rep gene. Right panel: samples were treated with DpnI digestion to remove all input plasmid; only AAV DNA that is rescued from the plasmid and replicated is detected. Plasmids pAAV-GFP and pAV2 were transfected in the presence (+ Ad) or absence of adenovirus. Hybridisation with the Amp probe confirms that all input plasmid was digested by DpnI treatment. The Rep probe detects replicated AAV DNA. M: monomeric replicative form; D: dimeric replicative form; larger replicative intermediates are visible above. Adapted from Bardelli et al., 2016.

Notes

Steps D3 to E5 should only be performed in areas designated for work with radioactivity. Please follow local directives.

Note on controls: several controls should be used when running this assay.

+ AAV, - adenovirus: no replication control. In the absence of adenovirus, AAV replication is absent or very low.

- Rep, + adenovirus: no replication control. Instead of wt AAV, use a replication-deficient AAV mutant or recombinant AAV vector (for example pAAV-GFP). This will also control for the absence of wt AAV contamination in the adenovirus batch used in the experiment.

The Amp probe is used to assure that input DNA is digested.

No DpnI control: allows the comparison of input plasmid between different conditions, and controls for the binding of the Amp probe.

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